1. Field of the Invention
The present invention relates to a process for releasing into aqueous medium polypeptides produced by yeasts and localized at least partially in the periplasmic space thereof. More particulary, it relates to a process for releasing into aqueous medium the lysozyme produced by yeasts genetically engineered for that purpose.
2. Description of the Art
The yeast Saccharomyces cerevisiae is increasingly used as a host for genetic manipulations aimed at producing polypeptides of commercial interest by fermentation. The increasing use of yeast is attributable to the various advantages that it shows over other industrial microorganisms, e.g., the fact that yeast is above all an alimentary organism. Another advantage of yeast is that its culture does not require absolute sterile conditions, so that it is particulary appropriate for large scale fermentations. In addition, its ability to live under anaerobic conditions makes it suitable in immobilized form for the continuous production of metabolites.
When using yeast for producing polypeptides, e.g., enzymes, it obviously is important that these be secreted through the plasmic membrane and excreted into the fermentation medium, from which they can then be recovered by using well known techniques such as adsorption or affinity chromatography. It is known that proteins secreted through the plasmic membrane of yeast tend to remain confined to the periplasmic space or at least to remain associated with the cell wall. This is often observed in yeasts of the Saccharomyces genus and particularly in the S. cerevisiae species (see R. SCHEKMAN and P. NOVICK, "The Molecular Biology of Yeast Saccharomyces, Metabolism and Gene Expression", J. N. Strathern et al. Eds, Cold Spring Harbor, N.Y., 1982, pp. 361-393). This peculiarity which may bring some functional advantage to yeast is, however, a major disadvantage from a practical standpoint when the production of proteins of commercial interest by fermentation is to be carried out. Indeed, in such a case, the benefit brought by secretion for recovering the protein of interest is lost since said protein, by remaining associated with the cells, must be separated from the whole of the cellular materials as in the case of an intracellular protein. This tendency of yeast to keep the proteins they secrete associated with their wall has been attributed to the fact that most of these proteins are heavily glycosylated. This is the case for invertase and for acid phosphatase which both contain a large proportion of polysaccharides comprising essentially mannose. One function of the glycosylated portion of these proteins would be to maintain their association with the polysaccharide matrix of the walls comprising essentially itself mannose (see J. O. LAMPEN, Antonie van Leeuwenhoek, 34, 1-18, 1968). According to this interpretation, the excretion into the medium of the .alpha. factor of Mat.alpha.-type yeasts or of the "killer" protein produced by some yeast strains can be explained by the fact that they are not glycosylated.
However, when non-glycosylated heterologous proteins are expressed in yeast, frequently only a fraction of the protein formed is excreted into the medium, even when it is equipped with a signal sequence allowing its secretion through the plasmic membrane. This is the case for human .alpha.1interferon (see A. SINGH et al., Nucleic Acids Res., 12, 8927-8938, 1984) the secretion of which was ensured by fusion of the corresponding gene with the DNA coding for the leader sequence of the precursor of the yeast .alpha. factor: only one-half thereof is found in the medium. This is also the case for chicken lyzozyme secreted due to its own signal sequence (see Belgian Patent No. 901,223). In such cases, it obviously is possible to recover only the soluble fraction of the protein but this would result in a loss of yield.
It also is possible to recover the fraction remaining associated with the cell, but this would require additional operations. Either solution would result in increased production costs which will reduce to some extent the above-enumerated advantages of utilizing yeast as a production organism.